A variety of biological and chemical assays have been developed for detecting the presence of compounds of interest in samples. In the biomedical field, methods for detecting the presence of specific nucleotide sequences, proteins or peptides are utilized, for example, in diagnosing various medical conditions, determining predisposition of patients to diseases, and performing DNA fingerprinting.
In general, biological and chemical assays are based on exposing an unknown sample to one or more known reactants and monitoring the progress or measuring the outcome of the reaction. It is often desirable to expose a sample to multiple reactants, to react multiple dilutions of a single sample with one or multiple reactants, to expose multiple samples to a single reactant, or to perform multiple repetitions of a particular assay for a given sample, in order to improve reliability. There is currently a high level of interest in the development of high throughput methods for performing multiple biological and chemical analyses of this type simultaneously, quickly, and conveniently.
One recently developed method for performing multiple chemical reactions simultaneously is to form a microarray of multiple spots of reactant molecules on a planar substrate such as a glass microscope slide, typically in a two-dimensional grid pattern, and apply liquid reagents and reactants to the slide to contact multiple spots simultaneously. Various reaction steps may be performed with the bound molecules in the microarray, including exposure of bound reactant molecules to liquid reagents or reactants, washing, and incubation steps. The progress or outcome of the reaction may be monitored at each spot in the microarray in order to characterize either material(s) immobilized on the slide or material(s) in a liquid sample. Although it is typical to immobilize known reactants on the substrate and expose an unknown liquid sample to the immobilized reactants and monitor the reaction between the sample and the various reactants in order to characterize the sample, it is also possible to immobilize one or more unknown samples on the substrate and expose them to a liquid containing one or more known reactants.
Microarrays are frequently used in analysis of DNA samples, but may also be used in diagnostic testing of other types of patient samples. Spots in microarrays may be formed of various large biomolecules, such as DNA, RNA, and proteins; smaller molecules such as drugs; co-factors, signaling molecules, peptides or oligonucleotides. Cultured cells may also be grown onto microarrays. For example, if it is desired to analyze gene expression by studying the presence of particular DNA sequences in a patient sample, the sample is exposed to a microarray of spots formed of oligonucleotides having sequences complementary to sequences of interest. If the DNA sequence of interest is present in a patient sample, it will hybridize with the bound oligonucleotides. The occurrence of hybridization at a particular spot then indicates the presence, and perhaps additionally the quantity, of the sequence associated with that spot in the sample. Hybridization can be detected by various methods. One commonly used method involves labeling the sample with a fluorescent dye, so that fluorescence can be detected at spots where hybridization occurred. Various types of slide readers are commercially available for reading microarray slides.
Microarrays offer great potential for performing complex analyses of samples by carrying out multiple detection reactions simultaneously. However, a current limitation of microarrays is the time and care required to process slides to obtain reliably high quality results. The need for high quality processing is particularly pronounced because individual microarrays slides are expensive and only limited quantities of the samples used in the reactions may be available, making it particularly important to obtain good results consistently.
Both manual and automated methods of performing microarray hybridizations have been developed. However, to date, no method has been completely satisfactory. In order to process a microarray manually, reagents or reactant solutions are applied to the microarray slide and a cover slip applied to spread the solution out into a thin layer that covers the entire microarray and prevents evaporation. Evaporation of solution and non-uniformity of the fluid layer are problematic. Moreover, the success of the procedure is largely dependent on the skill of the human technician. In addition, the hybridization fluid is static, which can limit sensitivity.
Various methods have been developed to overcome the limitations of manual slide processing. These range from simple slide processing chambers designed to simplify the application of solutions to microarray slides and reduce loss and leakage of solutions, to large and expensive machines capable of processing large numbers of slides simultaneously.
Loeffler et al. (PCT publication WO 00/63670, dated Oct. 26, 2000) describe a slide processing chamber designed for processing microarray slides. Freeman (U.S. Pat. No. 5,958,760, issued Sep. 28, 1999), Stapleton et al. (U.S. Pat. No. 5,922,604 issued Jul. 13, 1999), Stevens et al. (U.S. Pat. No. 5,605,813, issued Feb. 25, 1997) and Richardson (U.S. Pat. No. 6,052,224, issued Apr. 18, 2000) all disclose slide processing chambers not specifically disclosed for use in microarray processing, which however serve to illustrate the general state of the art relating to the processing of individual slides.
Devices capable of processing multiple slides simultaneously in an automated fashion are described by Custance (U.S. Pat. No. 6,238,910, issued May 29, 2001) and Juncosa et al. (U.S. Pat. No. 6,225,109, issued May 1, 2001).
All of the above mentioned patents or applications are incorporated herein by reference.
Devices for automated processing of microarray slides offer advantages in terms of reproducibility and ability to process large numbers of slides, but require relatively large sample volumes and are prohibitively expensive for labs that do not need to process large numbers of slides. Although reproducibility is significantly improved by automation, the results obtained with commercially available instruments may be of lower quality than those obtained with manual processing.
In many applications, it is desirable to mix or agitate fluid on the surface of the microarray during processing. In particular, if the microarray is used to detect materials that occur in low concentrations in the liquid sample, the amount of time needed for molecules in the liquid sample to diffuse to binding locations on the microarray may be a limiting factor. Some slide processing systems incorporate mixing functions but effective mixing in low volume hybridization chambers is difficult to attain.